Process for freezing erythrocytes



Oct. 17, 1967 A. P. RINFRET ETAL. 3,347,745 I PROCESS FOR FREEZING ERYTHROCYTES Filed'Deo. e. 1963 9 Sheets-Sheet 1 HEAT FLUX q/a AT BETWEEN THE SURFACE OF THE SOLID AND THAT OF THE BOILING LIQUID SECONDS THICKNESS OF VASELINE COATING IN MM,

A T TOR/VEI Oct. 17, 1967 A. P. RINFRET ETAL 3,347,745

OCESS FOR FREEZING ERYTHROCYTES 9 Sheets-Sheet Z v Filed Dec.

HEAT TRANSFER RATES IN BOILING LIQUID NITROGEN 0.05 MM. VASELINE comma 37.500 0.025 MM. I 37.500 3? BARE PROBE VASELINE COATING F m a m E. 6Q 25 3E MM. VASELINE COATING M. VASELINE COATING M. VASELINE COATING -1oo PROBE TEMP. IN

2 Q otdmxmziam -2oo C.

0.45 MM. VASELINE COATING 0.37 MM. VASELINE COATING CE 08? 3E o -i00 -2o0 PROBE TEMP. IN C.

lNVENTORS ARTHUR P. RINFRET CLEMENT W.COWLEY GERALD F. DOEBBLER ATTORNEY Oct. 17, 1967 A. P. RINFRET ETAL 3,347,745

PROCESS FOR FREEZING ERYTHROCYTES Filed Dec. 6, 1963 9 Sheets-Sheet 5 BOILING HEAT-TRANSFER RATES OF COATED AND UNCOATED CYLINDERS IN LIQUID NITROGEN VS,

' SPECIMEN TEMPERATURE 75 TO 200 MESH GROUND SUGAR ON GLYCERINE SLIGHTLY TRITURATED SUGAR ON GLYCERINE GLYCERINE HEAT-TRANSFER RATE IN THOUSANDS OF BTU/(HRXF T?) UNCOATED I 50 0 -so 400 45o -2oo SPECIMEN TEM P.C.

INVEN TORS CLEMENT W.COWLEY GERALD F. DOEBBLER A 7 TORNEY Get. 27, 1967 A. P. RINFRET ETAL. 393479745 PROCESS FOR FREEZING ERYTHROCYTES Filed D99. 6', 1963 9 Sheets-Sheet 4 IMPROVEMENT IN BOILING HEAT TRANSFER WITH SUGAR-GLYCERINE COATINGS VS.

SPECIMEN TEMPERATURE IMPROVEMENT IN HEAT TRANSFER E? I l I I I 50 O 50 -1OO -I5O -2OO SPECIMEN TEMP.C.

/N VE N TORS ARTHUR P.RINFRET CLEMENT W. COWLEY GERALD F'. DOEBBLER Oct. 17, 1967 A. P. RINFRET ETAL 3,347,745

PROCESS FOR FREEZING ERYTHROCYTES 9 Sheets-Sheet Filed Dec.

EFFECT OF COATING WEIGHT go EFFECT OF INSULATING FILM THICKNESS Uww Z OOEmQ UZIOOU .360 SEC.

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ATTORNEY f Oct. 17, I967 A. P. RINFRET ETAL PROCESS FOR FREEZING ERYTHROCYTES Filed Dec. 6, 1963 EFFECT OF INSULATING FILM 9 THICKNESS WATER TEMP. 4oc.

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OF BTU/HR. FT.

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EFFECT OF AGE All o I I I I I I' O 1 2 3 lNVENTO/PS ARTHUR P. RINFRET CLEMENT W. COWLEY Oct. 17, 1967 P. RINFRET ETAL 3,347,745

PROCESS FOR FREEZING ERYTHROCYTES Filed Dec. 6, 1965 9 Sheets-Sheet s INVENTORS ARTHUR P. RINFRET GERALD F. DOEBBLER CLEMENT W. COWLEY i 5U 5 53 El .55 54 XL {2 @Ct 17, 1957 A. P. RINFRET ETAL 3,347,745

PROCESS FOR FREEZING ERYTHROCYTES Filed Dec. 6, 1963 9 Sheets-$heet 9 x T f T T 5=:;

T I r r r f I T f lNVENTO/PS ARTHUR P. RINFRET 3 n I j CLEMENT w. COWLEY 54 m w I GERALD F. DOEBBLER A TTOR/VEV United States Patent C) 3,347,745 PROCESS FUR FREEZING ERYTHROCYTES Arthur P. Rinfret, Buffalo, Clement W. Cowley, Tonawanda, and Gerald F. Doebbler, Buffalo, N.Y., assignors to Union Carbide Corporation, a corporation of New York Filed Dec. 6, 1963, Ser. No. 333,786 14 Claims. (Cl. 167-74) tended preservation of stored blood.

The primary viable constituents in blood are the erythrocytes (red blood cells). Hence, the problem of preserving blood basically relates to the preservation of erythrocytes. The erythrocytes are globular in form and i.e., in the circulatory system of the body, have a life span of between about 100 days own supporting metabolism and outside the body it carries on its metabolic processes until the blood sugar is depleted and converted to lactic acid. As this occurs,

fused. This amount of degeneration is below acceptable levels, and it has proven impossible to stockpile such blood since each unit must be replaced every three weeks.

Experimentation with small quantities of whole blood, up to a few milliliters, in recent years has indicated that a much longer period of storage without progressive deis feasible at a sufliciently low temperature essentially stopped. It has that indiscriminant coolof erythrocytes in the temperature range .50 C. causes total hemolysis. Present analysis ascribes this hemolysis to the development of ice crystals in the extracellular spaces between the erythrocytes which withdraw pure water from solution and concentrate it between the erythrocytes. In effect, the erythrocytes are dehydrated and the concentrated intracellular solutes remaining, particularly salt, cause destruction such as the denaturing of protein, especially lipoprotein. The goal of prior experimentation with freeze-preservation techniques, therefore, has been to avoid erythrocyte injury by preventing the degradative biochemical eifects of high salt concentration concomitant with cooling the erythrocytes through the 0 C.50 C. temperature range.

One prior method of preventing such freeze-thaw hemolysis is to carry out the freezing and thawing at ultra-rapid rates on the order of hundreds of degrees C. per second such that insufiicient time exists for denature.-

order of 1 C. or less. At such high concentrations, how ever, the protective additives must be removed before the erythrocytes can be safely transfused. The

thus far barred the freeze-preservation of whole blood using glycerine for blood banking.

Another prior method of freeze-preservation involves the use of relatively low concentrations of sugar solutes as protective additives which may be intracellular such as glucose, or mixtures of intracellular and extracellular solutes) such as g1ucos and lacat rates intermediate the two aforeper second As m the aforementioned ultra-rapid freze-preservation technique, the physical heat transfer preservation technique would necessitate the use of a complex washing process as in the aforementioned slow freeze preservation technique if frozen and thawed blood in volumes larger than -a few milliliters were to be transfused. Consquently, the use of sugar solutes in freezepreservation processes is as yet not practical for blood banking.

Accordingly, it is an object of this invention to provide a system for preserving human erythrocytes in bulk quantities, meaning quantities of /21 pint and larger, which is free from the disadvantages of processes heretofore known. Another object of this invention is to provide a system whereby mixtures of human erythrocytes and polymeric protective additives can be preserved at low temperatures. A further object of this invention is to provide a process whereby such preserved mixtures may be transfused in the required quantities without removal of protective additives. These and other objects and advantages of this invention will be apparent from the following description, appended claims and drawings of which:

FIG. 1 is a boiling curve showing a plot of heat flux against the temperature difference between the surface of a solid and that of a boiling liquid;

FIG. 2 is a liquid nitrogen cooling rate curve in which cooling time is plotted against the insulating film coating thickness;

FIG. 3 is a series of heat transfer rate curves for various thicknesses of insulating film coatings;

FIG. 4 is a series of boiling curves of various insulating coating materials;

FIG. 5 is a boiling curve glycerine coating;

FIG. 6 is a series of cooling curves for sugar-glycerine coatings showing the effect of several variables over a temperature range of C. to -196 (1.;

FIG. 7 is a series of cooling curves for sugar-glycerine coatings showing the effect of several variables over a temperature range of 0 to 75 C.;

FIG. 8 is a series of warming curves for sugar-glycerine coatings showing the effect of the same variables as in FIG. 7 over a temperature range of 75 C. to 0 0;

FIG. 9 is a perspective view looking downwardly on a rectangular-type container for storing biological substances according to the present invention;

FIG. 10 is a perspective view looking downwardly on a novel cylindrical-type container for storing biological substances according to the present invention;

FIG. 11 is a perspective view looking downwardly on still another novel container for storing biological substances; and

FIG. 12 is a view of a longitudinal cross-section of the container shown in FIG. 11, taken along the line 12-12.

The following is a glossary of terms employed in the description of the invention and in the claims:

Red Blood Cell (RBC) Recovery-The in vitro amount of nonhemolyzed erythrocytes, expressed in percent, which remain after freezing and thawing; the measure of hemolysis being obtained by measure of the total hemoglobin of a sample of unprocessed blood as compared with the hemoglobin of the supernatant obtained by centrifugation of frozen and thawed samples.

Red Blood Cell (RBC) Survival-The in vivo amount for a specimen having a sugar- Oi frozen and thawed erythrocytes, expressed in percent,

which rernain in circulation in the blood stream; the measure of survival being obtained by radioisotope tagging of a sample of frozeinand thgwed test red cells before infusion and the subsequent eswtion of the tag level in the circulation; the degree of dilution of the tagged test cells in the circulation of the recipient being conTp 'ed with the degree of dilution of a control sample of u processed red cells previously injected in the same recipient.

Hematocrit-The volumetric ratio, expressed in percent, of blood cells (erythrocytes, leucocytes and thrombocytes) to the total volume of cells and cell-containing suspending medium.

Normal Hematocrit-the hematocrit of human whole blood, generally about 45%.

Isotonic Saline Solution0.85 gram sodium chloride (NaCl) in water to a total of milliliters; such solution exhibiting the same osmotic pressure as normal red cells.

Resuspension Stability-The in vitro amount of nonhemolyzed, frozen and thawed erythrocytes, expressed in percent, which remain after dilution in forty-fold excess, or greater, of isotonic saline or other medium; the measure of hemolysis being obtained by measure of the free hemoglobin of the supernatant obtained by centrifugation of a sample of frozen and thawed blood as compared with the free hemoglobin in the isotonic saline obtained by centrifugation of a diluted sample of frozen and thawed blood.

Efiiciencies of Process (EOP)Refer to erythrocyte recoveries after all losses during freezing, thawing, and resuspension are taken into account. Direct recovery times resuspension recovery equals EOP.

Concentration-The amount of an ingredient (usually a protective additive) in a medium (usually the suspending medium for the erythocytes) expressed as a percent (either by weight or by volume as indicated).

Final Mixture Concentration-The concentration of an ingredient (usually a protective additive) in the mixture including erythrocytes expressed as a percent computed arithmetically from the concentration of the ingredient in its medium and the relative quantities of the ingredientcontaining medium and the erythrocyte medium.

The prime objective of the present invention of providing a bulk blood preservation system which employs simple and rapid techniques, as would be required under large scale disaster conditions, requires that the compounds combined with the erythrocytes, to provide protection of the erythrocytes during freezing and thawing, be acceptable as transfusible substances; maintain the highest degree of erythrocyte osmotic stability, viability and functionality; and protect a very high percentage of the erythrocytes from lysis during the preservation process so that the amount of free hemoglobin and cellular debris does not exceed tolerable limits during transfusion of multiple units of preserved erythrocytes. It has been discovered that a high recovery of intact erythrocytes is achieved only when frozen in the presence of a medium containing a minimum of about 10% by weight of an extracellular protective additive; that osmotically stable cell suspensions are recovered only with extracellular protective additives which do not enter the cell; and that carefully controlled, rapid rates of heat transfer are necessary during freezing and thawing to get a high recovery of stable cells in the presence of extracellular protective additives.

An erythrocyte preparation with high recovery of intact cells on thawing and high stability on transfusion is provided by the present invention by the use of extracellular protective additives of the water soluble high molecular weight type such as polyvinyipyrrolidone (PVP), dextrose, and like polymers at a final mixture concentration between about 320% w./v. in combination wtih turbulent agitation during cooling and warming. Such substances do not penetrate the erythrocyte membrane and can thus be rapidly removed by simple centri fuging, Washing not being required, or left in the preserved erythrocytes without risk of large scale osmotic lysis during transfusion. Furthermore, due to their high molecular weight, their molar concentration in a recipients blood stream is considerably lower than prior additives and there is considerably less opportunity for the additive to effect the recipients blood stream than heretofore possible. These discoveries enabled the developmentaoi thflift lbasic processes which are particularly suited to the use 0 olyrneric additives.

The present invention provides a system for the freezepreservation of human erythrocytes in bulk quantities of transfusion unit size or larger by any of three basic processes, all of which involve freezing the erythrocytes in containers immersed in a low temperature bath and thawing them by immersing the container in a warm bath. Process I involves the freeze-preservation of whole blood, having its normal amount of erythrocytes, in the presence of an extracellular protective additive-containing medium. Process II involves separating the erythrocytes from whole blood such as by means of conventional centrifugation, and resuspending the erythrocytes in an extracellular protective additive-containing medium prior to their freeze-preservation. Process III involves the freezepreservation of the erythrocyte fraction of whole blood in the presence of part or all of its plasma and an extracellular protective additive-containing medium.

An essential processing parameter for all three of these basic processes is that the erythrocyte-containing mixture be turbulently agitated, such as by rapid shaking both during freezing, such that a solid shell of the frozen mixture is formed on the inner surfaces of the mixture container (called shell freezing), and during thawing. Agitation during processing has been discovered to minimize the dependence of freeze-preservation results on the mass thickness of the erythrocyte-containing mixture which has heretofore restricted prior methods to preserving mixture volumes of sufiiciently small cross-section such that the erythrocyte layer furthest from the mixture container inner surface is within about 5 mm. of such surface. With the present invention, larger mixture volumes merely require, in general, a greater degree of agitation to achieve acceptable results and the mass thickness of the frozen shell may be greater than about 5 mm. (up to at least about min.) without deleteriously affecting the results. Furthermore, the present invention minimizes the necessity of maintaining an absolutely uniform cross-section of the frozen mass which has heretofore been a requisite of prior freeze-preservation methods. The fact that a 5 mm. mass thickness is not critical when the mixture is turbulently agitated during freezing, but is critical when the mixture is stagnantly frozen, indicates that turbulent agitation contributes to the viability of the processed erythrocytes in some unexpected manner independent of heat transfer considerations.

Preferably, the rate of heat transfer between the refrigerant and the stored mixture is at least 14,000 B.t.u./ (hr.) (sq. ft.) and preferably at least 24,000 B.t.u./(hr.) (sq. ft.) during the entire freezing step so that freezing may be obtained in a sufficiently short period for high erythrocyte (REC) recoveries on the order of at least 90%. In order to obtain the desired heat transfer rate, the erythrocyte mixture storage container may be coated with a heat transfer promoting coating in the form of a thin insulating film having sufiicient insulating power to adjust the temperature difference between the heat rejecting surface of the coated solid and a boiling refrigerating medium to a value where more efiicient heat transfer will result as illustrated in FIGURES 1-8. The heat transfer rate may be further improved by applying a layer of powderous material on the previously mentioned thin insulating film which then serves as an undercoating as also illustrated in FIGURES 1-8. The powderous material layer is believed to further increase the heat transfer rate for at least two reasons; first, the discontinuity of the surface promotes bubble formation, and secondly, the exposed portions of the powder particles cool down rapidly whereby boiling in the nucleate regime is quickly established. Glycerine, for example, is admirably suited as a material for forming the thin insulating film, and finely divided silica, for example, has been found particularly effective in providing the powderous material layer. The undercoating and powderous layer may be applied to the container is filled with the The following materials in insulating film coating:

Mm. (1) Poxalloy adhesive 0.04-0.75 (2) Clear varnish 0.04-0.10 (3) Vulcanized rubber 0 04-023 (4) House parafiin 002-034 (5) Rubber paraffin 0 03-039 (6) Paper masking tape 0 30-090 (7) Rubber electricians tape 0 30-090 (8) Vaseline 0.01-0.95 (9) Asbestos 0.25-0.95 (10) Sodium silicate 0.11-0.17 (11) Kaolin 0.11 (12) Plaster of Paris 0.19

It has been found that the heat transfer rate between the refrigerant and the container Walls may be even further improved by applying a layer of powderous material to the thin insulating film coating. The powder layer provides a discontinuous surface for the promotion of bubble formation and the exposed portions of the powder particles cool down rapidly.

In addition to finely divided silica such as silica aerogel having agglomerate particle sizes of less than about 5 microns and crystalline zeolite molecular sieves A and X, both in agglomerate particle sizes of 2-4 microns, sugar powders are suitable. Silica aerogel, known commercially as Santocel, is preferred over sugar because it is more readily available in the ultra-fine sizes which are desired for best reproducibility in actual practice, while it may be necessary to grind sugar down to the correct mesh sizes in order to obtain an optimum coating.

It should be recognized that when a powder layer is to be employed, there is an additional requirement for suitable film coatings. That is, the undercoating must be formed of material which will receive and hold the powder. Both glycerine and an aqueous solution of polyvinylpyrrolidone have been successfully used. Glycerine has been employed alone and also diluted with methanol. Other suitable undercoating materials include oils in general, and the silicone fluids. A Water soluble undercoating is generally preferred so that it will wash 013? during the thawing step. Each of the above-mentioned materials is either water soluble or may be obtained in a water soluble form. For example, many detergent oils are available.

The solvent or diluent such as the methanol mentioned above can also be varied. For example, ethanol, water or any substance that will not impair the water stripping of the film may be employed. The function of the diluent is to adjust the properties of the insulating film (a) to produce a good adherent uniform film according to whether it is applied by spraying, dipping or otherwise, (13) to keep it fluid at least until the powder addition is made, and (c) in some instances, to act as a plasticizer and help prevent drying out and flaking off during the cooling step.

The thin insulating film and powderous layer may be applied to the outer walls of the erythrocyte mixture container in any convenient manner as, for example, by dipping the container into a solution containing the insulating material. In such case, the thickness of the insulating film may, for example, be controlled by adjusting the concentration of the solution and the number of dippings. However, the preferred method of applying the powderous layer is by spraying with a propellant gas onto the aged insulating film.

A refrigerant suitable for use in freezing the erythrocyte mixture must have a temperature of below about and relatively inexpensive. It also has an exceedingly low boiling point, namely 196 C. at atmospheric pressure. The liquid nitrogen employed can, for example, be obtaine-d by the well-known rectification of air. However,

other refrigerants may also be employed. Among those liquids which may be used are liquid air (containing normal amounts of nitrogen), helium, neon, argon, and krypton.

Liquid nitrogen and the other low-boiling refrigerants are saturated fluids at atmospheric pressure, and boil violently when a warm object such as the erythrocyte mixture storage container is plunged therein. The heat transfer is dependent upon the temparature difference (AT) between the fluid and the warm object as previously discussed. At very high values of AT, a vapor film is forced around the warm container resulting in very poor heat transfer. This vapor film becomes less and less stable as the AT is decreased and the heat transfer improves. At the AT of about 3 C. (for liquid nitrogen), maximum heat transfer is attained and drops off as the AT is reduced to zero. The application of the aforedescribed coatings of the container outer walls allow the surface in contact with the liquid nitrogen to be cooled very rapidly and provide a AT value closer to 3 C.

In the shell-freezing method, wetting of substantially all of the inner heat transfer surfaces of the container with the erythrocyte-containing mixture to form the solid frozen outer shell may be achieved by any convenient means which turbulently agitates the mixture. For example, a rapid reciprocal shaking motion may be imparted to the container while the latter is being chilled.

During the freeze-preservation of erythrocytes it has been found that the total time the erythrocytes spend in the temperature region between C. and 50 C. must be carefully controlled. This, of course, requires also the controlling of rewarming time after storage. The rewarming and subsequent thawing time can be materially reduced if the container is shaken while immersed in the warming bath. A convenient warming bath comprises warm water.

In the rewarming and thawing step, relatively cold container is as soon as the plunged into the warm water, a layer of thawed fiuid is formed contiguous to the container wall. Since this layer has a heat conductivity of about one-third that of the solidified substance and much lower heat conductivity than that of the container wall, it immediately reduces the heat-transfer rate to the stillfrozen portion of the mixture. However, the heat-transfer rate can be improved if the layer of thawed liquid is agitated. If the container is shaken during the warming and thawing step, relative motion is imparted to the thawed liquid and the solid shell which, in turn, improves the heat-transfer rate across said liquid layer.

To minimize heat-induced hemolysis of the erythrocytes, the temperature of the thawing is generally maintained at 37 C., but bath temperatures as high as 55 C., have been employed successfully.

While most quantities of preserved erythrocytes are intended for in vivo uses, the utility of the instant process is equally well applicable to in vitro uses of erythrocytes. An important example of applications of the latter type is the use of red blood cells for serological work in identifying anti-bodies in patients who may have transfusion reactions. For this work it is advantageous to have on hand a convenient supply of samples of erythrocytes of all types.

In order to meet the important requirement that thawed erythrocytes be transfusible with little or no post-thaw processing, it was found necessary to use a protective additive that does not enter the cell. Red cells in the presence of intra-cellular additives such as glucose, glycerol, or dimethylsulfoxide, at concentrations that afford protection, lyse immediately on infusion. A principal problem, then, was the selection of a protective additive that does not enter the red cell, affords protection during freezing and thawing, and is pharmacologically acceptable on infusion. Of the many materials tried, hydrophilic polymers and particularly polyvinylpyrrolidone (PVP) appeared most promising. PVP resulted in the highest recovery of intact cells capable of surviving to a high degree on infusion, and had been used for many years as a plasma volume expander without manifestation of acute effects. Dextran, human serum albumin and Haemaccel, although physiologically acceptable, did not give the same degree of protection.

Among the matters studied have been:

(1) Red Cell Stability (a) in vitro (b) in vivo (2) Processing Variables (a) Protective additional concentrations (b) Freezing and thawing conditions Details of these studies are given in the following sections.

PROCESS I Whole blood modified by addition of polymers such as PVP, Dextran, albumin, and the like to a final mixture concentration of 5-15% w./v. withstand rapid bulk freezing and thawing with the least alteration, the highest RBC recovery, and the greatest in vitro stability after thawing. Preferred final mixture concentrations of the protective additive are 610% w./v. and should not be greater than about 20% w./v. Above a polymeric protective additive final mixture concentration of 20% w./v., osmotic stability of processed erythrocytes is substantially reduced rendering the erythrocytes unsuitable for transfusion.

The whole blood can be obtained by conventional procedures, for example it can be drawn from a donor in one pint quantities into a suitable nontoxic anticoagulant medium such as citrate-containing anticoagulants, heparin, ethylene diamine tetraacetic acid (EDTA), acid citratedextrose (ACD), and the like. Coagulation of blood may also be avoided by defibrination of the drawn blood or by the removal of the contained calcium from whole blood by suitable ion-exchange techniques.

(A) DESCRIPTION OF PROCESS In a preferred embodiment of Process I, 420 cc. of blood is drawn into cc. of a solution containing 45% ACD-A and 26.5% PVP (Plasdone C) to give a total volume of 580 cc. and a final PVP concentration in the mixture of 7% w./v. The container is aluminum which is corrugated to give a high ratio of surface to volume, and has a volume of 1000 cc.

The container of blood is coated with a thin (0.004 in.) heat transfer-promoting layer of PVP and then immersed into liquid nitrogen while being rapidly shaken. This results in the formation of a shell of frozen blood 3-9 mm. thick. After immersion for 120 see. the container is stored at C. or below until needed.

Thawing is accomplished by shaking the container in a bath of water at 45 C. for about 75 sec., after which it is immediately withdrawn and stored at 4 C. until administered.

(B) STABILITY OF CELLS (1) IN VITRO MEASUREMENTS (a) Recovery When red cells are frozen and thawed in the presence of plasma plus PVP, some of the cells lyse. The extent of lysis is determined by the concentration of PVP and the thermal regimen experienced by the cells. Under best conditions, 97% of the cells are reproducibly recovered intact, as measured by the loss of hemoglobin from cells. Potassium ion loss is greater than that resulting from lysis alone and is in the order of that of 2l-day-old bank blood; being 22 meq./l. vs. 23 meq./l. for 21-day-old blood. Direct infusion of such a product introduces free hemoglobin and potassium ion into the circulation in addition to the PVP required for protection. This is discussed further in the next section.

(b) Resuspension Stability Loss of hemoglobin and potassium from cells is a direct manifestation of cell damage. There is additional damage which is shown by the loss of cells on infusion and which can be demonstrated in vitro by diluting cells with isotonic saline. There is additional hemolysis, the extent of which can give an indication of the degree of trauma experienced by the cells. Preparations that give 97% recovery of intact cells on thawing may lose an additional to 17% of the cells on resuspension in fortyfold excess (or greater) of isotonic saline. Those that show the largest additional hemolysis have the poorest survival on infusion, and those with small loss may, or may not, have good survival.

It appears, then, that in vitro tests do not distinguish unequivocally between preparations that will show the best in vivo survival and those that are poorer, but if they indicate a preparation is less stable, lower survivals will be obtained with it.

(2) IN Vrvo MEASUREMENTS (a) Survival After Infusion (b) Mechanism of Loss of Red Cells Following Infusion Immediately When intact cells disappear after infusion, they may be lysed in the circulation or may be removed in the RE system. Evidence against any marked lysis is given in Table I-2, which tabulates data obtained in one-pint transfusions. Column 8 represents the percentage of the total infused hemoglobin that is not accounted for as free hemoglobin in the plasma. It is equivalent to the recovery of intact cells after transfusion. Comparison (Column 9) of this value with the recovery of the cells before transfusion (Column 1) shows that stable cell preparations, as indicated by saline resuspension values of 87% or greater (Column 2), have lost less than 2% of their cells b3 lysis on transfusion. Although there are uncertainties in these measurements it is unlikely that much free hemoglobin has been removed from circulation in this time, for the haptoglobin-binding capacity has not been exceeded.

It would appear, then, that red cells frozen and thawed in the presence of plasma plus PVP are not removed primarily by lysis in the first min. after transfusion stable preparation might well lose some of the population by intravascular lysis in the period immediately after transfusion.

TABLE I2.ONE-PINT TRANSFUSIONS [Whole blood PVT] Using the optimal combination of processing parametersPVP concentration, freezing and thawing rates, container size and geometry described abovean average of of the cells been found circulating 24 hrs. after transfusion. Representative data are given in Table I-l.

TABLE I1.AVERAGE IN VIRO SURVIVAL OF FROZEN A 6 7 8 9 Plasma Total mt. of Postemo- Circu- Saline Volume Hemo- Transglobin lating 160 Normal Yield Transglobin fusion 30-Min. Hemo- (1 Hgb [(1) (8)] Recovery, (1:100), fused, Trans- Plasma Postglobin circ.) Percent Percent ml. fused, Vol., Trans 30-Min. Hgb in g. ml. mg.- Postfused Percent Trans,

96.8 88. 7 386 35. 5 2, 558 68 1. 74 95. 1 1. 7 95. 5 84. 1 390 38. 2 2, 678 19 2. 44 93. 6 1. 9 95. 6 86.8 381 34. 2 2, 740 49 1. 34 96. 1 -0 96. 5 88. 9 377 34. 3 2, 698 59 1. 59 95. 4 1. 1 97. 3 90. 3 382 33. 6 3, 048 30 0. 91 .97. 3 0 97. 1 89. 9 0 97. 5 92. 1 379 33. 4 l, 904 35 0. 67 98. 0 -0 97. 1 90. 8 383 39. 1 2, 333 48 1. 12 97. 1 0 96. 2 87. 5 0 97. 3 89. 4 269 24. 2 2, 197 37 0. 81 96. 7 0. 6 96.5 85.0 395 g 38.0 2, 639 2.50 93. 4 3.1 95. 5 85. 7 386 38. 2 2, 819 84 2. 37 93. 8 1. 7 96. 8 86. 5 387 37. 9 2, 462 71 1. 75 95. 4 1. 4 96. 8 88. 5 384 34. 9 2, 601 66 1. 72 95. 1 1. 7 97. 2 87. 6 384 38. 4 2, 465 70 1. 72 95. 5 1. 7 96. 3 84. 3 380 36. 4 2, 467 77 1. 9O 94. 8 1. 5 95. 1 81. 6 396 37. 6 2, 860 70 2. 00 94. 7 0. 4 96. 3 85. 0 391 37. 2 1, 767 87 1. 54 95. 9 0. 4 96. 1 85.1 390 41. 8 2, 384 88 2. 10 95.0 1. 1 96. 0 85. 7 395 39. 9 2, 327 99 2. 30 94. 2 1. 8 95. 8 83. 7 396 41. 9 2, 558 100 2. 56 93. 9 1. 9

(c) Evaluation of Processing Parameters When extra-cellular, polymeric protective additives, such as PVP, are used, stringent control of processing conditions is needed to give the highest recovery of stable cells in reproducible fashion. An important part of recent ND THAWED BLOOD studies has been the determination of optimum processing conditions and the degree of variability permitted, using in vivo survival as the measure of stability. Variation in mixture volume from 10 to 60% of container capacity indicated that red cell recovery is sensitive to volume only below about 30%.

(1) PVP concentration-The minimum final mixture concentration of PVP which gives very high stability of red cells has been found to be 7 g. per 100 ml. (7% W./v.) final red cell suspension. Variation in the freezing and thawing conditions have not resulted in the preferred use of a lower concentration.

(2) PVP molecular weight-All recent preparations evaluated by clinical assay have been made with Plasdone C, K-30 PVP. This has an average molecular weight of 40,000. It has been established that PVP K25 (from Light and Co.) and PVP K-22 (from Antara Products), both of about 25,000 average molecular weight, give cells of comparable stability as measured by in vitro tests (Table I-3).

TABLE Lil-Th5 VITRO STABILITY OF RED CELLS PRO- ECTED BY VARIOUS PVPS PVP was present in whole blood-ACD at a concentration of 7% w./v. Fifty-milliliter samples were processed under identical conditions.

(3) Freezing cnditi0ns.-Strict control of the freezing conditions continues to be necessary for reproducible attainment of high recovery of stable cells. Immersion of blood containers in a 500-centistoke solution of PVP K-30 in methanol gives a coating that results in optimal heat transfer. Faster or slower cooling than is obtained with this mixture gives poorer results. Shaking in liquid nitrogen during freezing to give a shell of frozen blood has proved to be necessary, for in no test without shaking was a high recovery of stable cells achieved.

(4) Thawing conditions-Shaking of a container of frozen blood in a warm water bath to thaw gives best results.

Collection of blood.-Blood has been collected in glass bottles or plastic packs and then transferred to the metal container or collected directly into the metal container without apparent difference in results. Blood has been added to a mixture of ACD and PVP, or PVP has been added to blood and ACD ,again without noticeable difference.

There have been differences noted among the recoveries and osmotic stabilities after freezing and thawing of bloods collected by various agencies (Table 1-4). There has been no apparent difference in the techniques of collection. It is hypothesized that the difference results from the time of collection and, more likely, from the fasting requested of the Agency 1 donors. The first clinical test of blood from donors who have fasted indicates this may be a factor in getting a somewhat more stable cell.

TABLE I3.VARIATION OF RED CELL STABILITIES AMONG COLLECTION SITES Pints (420ml. blood plus lfiO-rnl. ACD-PVP) were collected, frozen, and thawed under identical conditions;

12 PROCESS n It is well established that both the composition of the suspending medium and the conditions of cooling and warming influence the injury and protection of red cells during freezing and thawing. Using artificial media of defined composition it is possible to study the effects of each and all constituents in which the red cells are suspended during processing. Moreover, the autologous plasma is available for subsequent resuspension of the thawed red cells. Their in vivo viability can be estimated under conditions equivalent to whole blood control specimens.

Using red cells suspended during freezing and thawing in defined media eliminates many restrictions on composition. Optimum chemical conditions can be readily sought for minimizing injury. The use of extracellular polymeric protective solutes allows rapid removal after thawing and immediate resuspension without osmotic problems inherent with small molecular weight solutes. Simple conventional centrifuging sufiices to separate the red cells from their suspending medium and washing is not necessary.

Shell freezing is employed, this being accomplished by mechanical agitation at controlled frequencies in liquid nitrogen. Cooling rates are varied by varying the mixture volume and the use of appropriate heat transfer promoting coatings on the outside surfaces of the containers.

Thawing is accomplished by mechanical agitation (to promote convective heat transfer) in warm Water. Conditions for particular experiments are described in foot notes to each table.

For experiments described in this section (i.e. Process II) whole blood-ACD was separated by centrifuging into the cellular and plasma fractions. The red cells were resuspended in polymer solutions and subjected to freezing and thawing. Best results occurred with final mixture concentrations of the polymeric protective additive of 10- 20% w./v. Direct recoveries and resuspension recoveries of red cells were estimated as described above.

(A) CHEMICAL PARAMETERS- Polyvinylpyrrolidone K-30 (average M.W. 40,000) has been studied most extensively since preliminary small volume tests indicated its superiority over a variety of other polymers including dextran, gelatin, oxypolygelatin, low molecular weight (M.W. 10,000) polyvinylpyrrolidone and others, although these are nevertheless superior over other non-polymeric protective additives. Table IL-l shows the results of varying PVP concentration in a system containing red cells suspended in an equal volume of PVP in isotonic saline. Similar studies using K-lS (M.W. 10,000) PVP gave recoveries ranging from 69- over the same concentration range. Direct recoveries increased continuously with concentration, however, resuspension stability passed through a distinct optimum.

TABLE ILL-EFFECT O'F CONCENTRATIONS OF POLY- VINYLPYRROLIDONE ON RED CELL RECOVERY aluminum con- Thawed at 45,

at the concentrations shown in 0.15 M NaOl. Frozen in tainer using PVP-MeOH coating, 200 c.p.m. agitation. 200 c.p.m. agitation.

Salt concentration influenced direct and resuspension recoveries. Both the absence of salt and its presence at 0.15 M were less favorable than an intermediate low concentration (Table II2).

TABLE II2.EFFECT OF CONCENTRATIONS OF POLY- XgzlgIgYRROLIDONE AND SALT ON RED CELL RE- Salt on Red Cell Recovery Polyvinylpyrrolidones of K-values ranging from about 12 to 30 were examined. Molecular weights above about 10,000 were required for optimum red cell recovery.

(B) PHYSICAL PARAMETERS Optimum coating heat transfer conditions were obtained by coating the metal container with a solution of polyvinylpyrrolidone K-30 in methanol (500 centistokes viscosity).

Thawing by mechanical agitation in Water at 55 im proved in vitro direct and resuspension recoveries by several percent as compared with results obtained at 45 C.

Variation in mixture volume from to 60% of container capacity indicated that red cell recovery was sensitive to volume only below about 30%. This could be ascribed to excessively rapid cooling or altered formation of the frozen shell during cooling.

Plastic containers were inferior to metal in terms of direct recovery. Resuspension stability was similar (Table II-3).

TABLE II3.COMPARISON OF METAL AND PLASTIC CON- TAINERS FOR PRO CESS II (PVP K-30 20%0.05 M N aCl) Direct Resuspension Recovery, Percent Percent Container RBC Recovery Saline PVP container of rectangular cross-section 64 x 64 x 19 mm.

B Aluminum Capacity 75 ml.

b Low density polyethylene bottles of rectangular cross-section 52 x x 24 mm. Capacity ml.

Each type container filled to 45% of capacity and frozen with mechanical agitation in liquid nitrogen and thawed in 45 0. water.

(C) PROCESS DEVELOPMENT Table II-4 shows results obtained in eight experiments using 15% PVlP-0.05 M NaCl as the meduim. Table II-5 shows the variations in composition of the medium. Essentially equivalent results have been obtained in bottles and in plastic bags (Table II-6). The use of plastic packs allows considerable improvement in processing time since centrifugation can be done at high speed. Approximately 15-20 minutes are required to separate the thawed cells.

TABLE II- 4.PROCESSING OF PINT VOLUMES OF BLOOD BY PROCESS II Percent Percent Resuspension Direct Recovery Exp. No. RBC

Recovery Dextran a Plasma a Resuspension of RBC Gentran M.W. 72,000 approximately).

RBC suspended in equal volume 15% K-30 PVP0.05 M NaCl. Frozen in pint aluminum container (capacity 1,100 m.l approximately) with PVP-MeOlI coating, 200 c.p.m. agitation. Thawed 25 seconds, 200 c.p.m.,

wa er.

1 Volume 01 blood diluted IOO-fold with physiological saline then allowed for free hemoglobi b Thawing at 200 c.p.m. in 45 0. water bath.

Blood rozen: 300

R Additive Composition Percent Intact Cells Number Thawed Blood Reconstituted Blood PVP NaCl Glucose of Tests (percent) (M) (percent) Direct RBC Saline a Direct RBC Saline e Recovery EOP Recovery EOP 20 05 0 b 4 90 81 95 88 20 05 0 13 94 81 95 88 20 075 0 1 96 82 96 84 20 10 0 1 96 76 91 79 20 .15 0 3 94 88 71 15 05 0 3 93 96 15 075 0 1 77 93 79 20 05 2 5 96 86 98 88 to stand )6 hour prior to analysis Other samples were agitated at -160 c.p.m.

cc. of additive per unit of packed cells from a pint collection.

Freezing Conditions: 200 c.p.m. in liquid nitrogen using corrugated aluminum 1 pint contaners.

TABLE II6.EFFECT PRESERVATION WITH PROCESS II Percent Intact Cells Anticoagulant [N201] in Thawed Blood Reconstituted Additive Blood gjg g @733? Egg gg; Clinical studies of Process 11 have served a several- Recovery Recovery fold purpose. Most important, a high in vivo viability of human red cells subjected to rapid freezing and thawing .05 eae 75 89.6 82.5 while protected by a polymeric solute is unequivocally 'l as e2 demonstrated I1 9319 7415 87:7 7827 Results of transfusions of half pint volumes of blood 2% 3%; 7,21% 2%; $323 frozen by Process 11 are shown in Table lI-7. Similar .05 94.2 81.9 95.7 90.9 results for full pints are shown in Table 11-8. .05 94.1 80.6 95.5 90.4

Additive Solution: Plasdone-C solution containing NaCl. 300 cc.

added to each unit of packed cells.

Containers:

Reconstitution: and resuspend in autologous plasma.

No heat transfer-promoting coating. Thawing Conditions: 150 c.p.m. agi

tation in C. water bath.

Remove supernatant from packed cells after thawing TABLE II-7.SUMMARY OF HALF-PINE RESULTS Percent Recovery Percent Survival 11 Conditions No.

Items Direct Resp! 26 hr. 24 hr. 48 hr. 72 hr.

Frozen Proc. II, 15% PVP--LS 943:1 98:4:13 86x13 i 79112 74:l:l1 Frozen Proo. II, 20% PVP-LS 905:2 873:9 733:8 67111 68ziz8 Frozen Proc. II, 20% PVP-NS 88:1:3 85:1:8 755:9 733:7 715:9 Frozen Proc. III 90 79 73 73 Controls, unfrozen fresh- 103i? 97:1;8 92:1;8 905:9 Controls, 4-day, 4 C. storage 105 103 98 97 a Resuspens ion recovery =percent recovery following resuspension 0i thawed cells in plasma.

b Survival by consecutive Chromium-51 procedure; percent of transfused BBC.

0 Equal volumes RBCs and additive solution: LS=0.05 M NaGl; NS=015 M NaCl.

Process III=equal parts plasma and 30% PVP; approximately 2 vol. plasma-PVP medium used for each Volume RB C.

Standard deviations shown as ivalues after each average value.

TABLE II8.RED CELL SURVIVAL FOLLOWING TRANSFUSION OF FULL PINTS OF BLOOD FROZEN BY PROCESS II Volume Direct Resus- Percent RBC Survival Transf. No. Transfuscd BBC pension (rnL) Recovery, Recovery;

percent percent 30 min. 24 hr. 48 hr. 72 hr.

A. 15% PVP-LS:

785:7 l 765:6 7514 Control 01 501 99 98 Control C2 508 76 72 74 Volume Direct Resus- Percent RBC Survival Transf. No. Transfused RBC pension (ml. Recovery, Recovery,

percent percent 30 min. 24 hr. 48 hr. 72 hr. D. 20% PVP-LS:

Avg. :1: S D 74i6 64:|:5 57:]:4 5515 Control D1 Control D2 Equal volumes container. Thawe Controls: LS: 0.05 M

(D) PVP-HUMAN SERUM ALBUMIN The presence of insoluble B-lipoprotein in suspensions of cells containing plasma and PVP led to test of cell suspensions containing less, or no, plasma. Blood was centrifuged to pack the red cells and the supernatant plasma removed. Red cells were then suspended in a medium containing PVP and albumin. Because we had observed, when testing serum albumin as a protective material, that it increased the protective action of PVP when red cells were processed in a solution containing both, we have concentrated on this mixture.

Processing involves separation of red cells from plasma, washing by resuspension of the cells in isotonic saline when the plasma is to be substantially completely removed; separation of the red cells from wash solution and resuspension in a solution containing PVP and albumin.

or red 0 d by agitation in 45 C (a) Description of Process Blood (450 ml.) is collected into 72 to 75 ml. of ACD-A and then equal volume of additive containing 14% Plasdone C, 3% human serum albumin, and 0.6% sodium chloride. The suspension is frozen in the same way as whole blood plus P (a) In Vitro Stability (c) In Vivo Stability Assay of survival of cells following infusion showed the cells to be more stable-than those recovered from the blood plus PVP. The loss of cells 24 hr. after transfusion was only 12%, in contrast to the loss of cells from the whole blood plus PVP (Table II-9). This compares favorably with the survival (93 to 95%) of fresh cells that have not been frozen and thawed.

TABLE II9.PVP-ALB UMIN PROCESS Survival Amount Trials Recovery Processed 30Min. 24Hr. 48 Hr. 72 Hr.

%pint 47 97110.3 87 -i=6 88.117 823:? 785:7 Pint 5 97.3i0.3 891:5 853:5 783:7

Blood collected into ACD-A. Cells separated and resuspended in 14% P, 3 albumin and 0.6% NaCl. Thawed cells separated from PVP solution and resuspended in autologous plasma for transfusion.

(d) Processing Conditions PROCESS III plasma and polyvinylpyrrolidone Table III-1 improvement in red pension recoveries resulted when plasma was present.

When the amount of plasma was'varied from 0 to 70% of the medium (by volume) at constant PVP concentration (15%), direct red cell recovery was observed to vary only slightly, while a marked improvement in saline and PVP (isotonic-isooncotic resuspension stability resulted (Table III-2). Above a level of 50% plasma, no improvement was found.

These observations suggested a process in which only a part of the plasma would be removed from whole blood prior to combining with an extracellular protective additive solution. This method has been designated Process III.

(A) CHEMICAL PARAMETERS solution. As shown in cell direct and resus- TABLE III-L-EFFEC'I OF VARYING THE COMPOSITION OF MEDIUM FOR PROCESS II 1 K-30 PVP in 0.05 M NaCl.

53 m1. of RB C-PVP or RBC-Plasma-PVP mixture frozen in aluminum containers coated with PVP-MeOH by agitation at 200 c.p.m. 2% inch amplitude in liquid nitrogen and thawed at 200 c.p.m. in 45 water.

TABLE III-2 Percent Plasma in Direct RBC Percent Resuspension Suspending Medium Recovery Stability Saline RB C in equal volume PVP K-30 in 0.15 M NaCl. Frozen in aluminum container using PVP-MeOH coating, 200 c.p.m. agitation. Thawed at 45", 200 c.p.m. agitation.

TABLE III3.-EFFECT OF POLYVINYLPYRROLIDONE CONCENTRATION IN PROCESS II K-30 PVP Cone.

Resuspension Recovery, in the suspending Direct Percent RBO Perceu medium w. Recovery Percent Saline PVP I RBC/Medium=1:2. Medium: 50% Plasma-PVP-O.1 M NaCl.

Varying the ratio of red cells to suspending medium improved direct red cell recoveries and resuspension recoveries. The presence of l to 2 volumes of medium (50% plasma-15% PVP) per volume of red cells gave recoveries significantly better than lesser volumes (Table 111-4). Larger volumes of suspending medium were equivalent but did not improve recoveries.

TABLE III-4.EFFECT OF VOLUME RATIO BETWEEN RED CELLS AND MEDIUM (PROCESS III) Percent Resuspension Recovery Vol. Medium Direct Percent Per Vol. R130 R130 Recovery Dextran Saline b PVP b e Dilution 1:2.

b Dilution 1:100.

RBC separated from whole blood washed with plasma-PVT, and resuspended in volumes of medium shown per volume cells. Frozen in aluminum container with PVP-Methanol coating with mechanical agitation 200 c.p.m. Thawed at 45 C. and 150 c.p.m.

Plasma, polyvinylpyrrolidone, and salt concentrations were studied simultaneously to define an optimum composition and delineate the useful limits of this system. Representative results are shown in Table III-5. An optimum in PVP final mixture concentration of about 13% w./v. exists. Plasma concentration can be as low as 15-30% of the medium by volume. Salt appears to be injurious at 0.15 M but at lower concentrations can be varied considerably without affecting red cell recovery.

TABLE III5.EFFECTS OF COMPOSITIONS OF THE LIE- DIUM ON RED CELL RECOVERY OBTAINED BY PROC- ESS III Concentration of Salt in Additive Percent Percent Plasma a PVP b 0.0 lVI 0.05 M 0.15 M

Vol. percent plasma in medium. W./v. percent K-22 PVP in medium. 6 NaCl cone. (molar) in PVP soln. added-Does not include salt from plasma. (See Text).

Values are percent percent resuspension recoveries in isotonic saline (1:100). All samples consisted of RBC (1 vol.)+medium (2 vol.). Frozen in uncoated alummum containers in liquid nitrogen at200 cum. and thawed in 45 water at 150 'c.p.m.

direct RBG- recovery; values in parentheses are Albumin at a final mixture concentration of 3-5% was observed to effectively substitute for plasma. The complete absence of salt led to reduced red cell recovery. However concentrations of only'0.02 M or above ap- 5 peared sulficient.

(B) PHYSICAL PARAMETERS Excessively rapid agitation, especially during thawing, reduced red cell direct and resuspension recoveries (Table III-6). Using mechanical agitation at an amplitude of 2 /2 inches, an optimum frequency of approximately 200 cycles/minute was observed. Thawing can be carried out at low frequencies of agitation and even manual shaking (approximately 75-100 cycles/minute) is adequate (Table 111-7). Variation in mixture volume, from 10-60% of container capacity indicated that red cell recovery is sensitive to volume only below about 30%.

Values are percent Direct RBC Recovery.

Values in parentheses are percent Resuspension Recovery in Saline. BBC n equal volume of 50% plasma-% K-30 PVP-OJ. M NaCl. Frozen in aluminum containers with PVP-MeOH coating, variable a agitation, in liquid N Thawed in 45 C. water, variable agitation.

TABLE III7.EFFECTv 0F AGIIATION CONDITIONS DURING THAWING 'ON PROCESS III Agitation Percent Resuspension 40 Frequency Direct Recovery Cycles/Min. RBC

(Thawmg) Recovery Saline PVP Manual '(75100)- 96 88 94 96 85 92 95 87 93 96 88 94 BBC in equal volumeof 50% wJv. plasma-15% w.-/v. K30 PVI 0.1 M NaCl. Froaen in aluminum containers with PVP-MeOH coating 200 c.p.m. agitation, in liquid nitrogen. Thawed in 45 0. water.

Red cell recoveries decline whenthawingis carried out-in water at temperatures below 30 C. Equivalent recoveries were obtained when thawings. were done in water at 30 to 45 C. (Table 111-8).

TABLE III8.-EFFECT OF TEMPERATURES OF THAWIN G ON PROCESS III RECOVERY" 60 Thaw Bath Direct Percent Percent Recov- Temperature, RBC Recovcry in Dextran O. my

RB'C/Medium= 1:1; Medium: plasma-15% PVP0.1 M NaOl.

Although more sensitive to suchfactors as low concentrations' of plasma'or polyvinylpyrrolidone, samples frozen and thawedin polyethylene containers gave in vitro recoveries-equivalentIto those obtained in aluminum containers of approximately equal geometries (Table III-9).

shown in Table III-ll. Precision has 14% in all cases. Accuracy evidenced by control been approximately appears to be very high as cell survival of 99:4% immediately Direct Percent Resuspension Recovery Percent following infusion as compared with the theoretical level Container Recovf 1007 Saline PVP For Process HI, optimum conditions in terms of red cell survival appear to require metal containers for freez- 32 3 2 ing. Thawing at 37 C. was better by several percent than thawing at or Polyvinylpyrrolidone final mixture concentration between 10-15% w./v. plasma coni l l0 s i See Oomote Tab an 3 centration between 15 and and salt concentration PROCESS DEVELOPMENT between 0.055 and 0.10 molar afford essentially equivalent From the standpoint f civilian r military medicine preparations onthe basis of in VlVO red cell survival. Diluunder conditions where separation and resuspension of tlon heat tl'ansffir condlfilonS $tud1ed fiPP to red cells before transfusion would not pose a serious 15 affect sllghtly Immediate post-mfuswn survlvai, u not limitation, Process 111 alfords a useful means of preserva- 24410111 survlvaltion when polymers are used as protective additives.

Table III-10 shows results obtained in processing full units of blood. The high dilution systems involved adding TABLE III-lL-CONIROL STUDIES or RED CELL PVP to whole blood without removal of plasma. Volumes 20 SURVIVAL up to almost of container capacity (up to 950 ml.) have been frozen and thawed with no reduction in re- PercentRBO P No. Recovery Survival covery of red cells. Conditions Tests TABLE III10.-PROCESSING OF PINT VOLUMES OF BLOOD Direct I Resusp. 10-30 min. 24 hr.

Dilution Percent Percent Resuspension Recovery Untreated Blood 10 995:4 9114 Exp. RB O/Total Direct Prefreeze Handling 5 99 =b3 97=l=3 N 0. Vol RBC Recovery Dextran B Saline b I PVP b Autologous blood, collected by venipuucture, labeled with Gr 0 hr.) end-reinfused. 1 112-6 95 86 93 Processed according to Process III (in 50% Plasmal5% PVP-0.1 2 1:2. 6 95 96 M NaCl) including all transfers, shaking (200 c.p.m.), resuspension in 3 1:2. 6 96 96 83 6 dextran, etc., but without freezing and thawing. 4 1:2. 5 d 94 95 87 93 5 1=2.5 97 98 so 91 6 1:4.1 96 9s 88 91 7 96 98 TbBLE III12.EFFECT OF THAWING BATH 0 TEMPERATURE ON RED CELL VIABILITY (PROCESS III) 2 it t i 38 tits esuspen e e Resuspension 1:2 in 5% albumin-saline. g gzg gifg gi d Incompletely thawed in 45 seconds. Thaw Bath N0 s R Slfipendtit; 507? 1w/v. plasma-l5Z2wJv. PYP-OJ M NaCdl Temp 0 O Testis in re. ioss own. a ioso .52.6 were sys ems wi p asma remove followed by reconstitution to volume with PVP solution. Ratios of 40 Direct. Resusp' 10*30 24Hrs' 1:4.1-4 7 were systems with PVP solution added directly to whole blood giving volumes of 800-900 ml. at time of freezing All frozenin PVP-MeOH 3 962% 97 i1 ssifi 715:5 gogted pint atlutmmunil conauers (capacity 110% fol oapproigm zi tiely) at 6 945:2 97 i1 933:6 781:5 0 c. .m agi a 1011 in iqui m rogen approxlma e y9 secon s awmg in 45 water, c.p.m. 45-50 seconds. 3 865:6 963:1 8km 693:3

Process III has been studied clinically in a series of small volume (10 ml.) transfusions reported in Tables. III-l2 through III-l7. Immediate (IO-30 minutes) and 24 hour survival for 10 untreated control samples and 5 samples processed without freezing and thawing are System: BBC in suspending medium containing 50% w./v. plasma- 15% w./v. PVP-0.1 M NaCl.

Dilution: BBC/Total volume=1/2. 6.

Conditions: 50 ml. in aluminum container coated with PVP-MeOH. Mech. agitation in liquid N; 200 cycles/min. (c.p.m.) 2% in. amplitude. Thawed at 2545 0., 150 c.p. In.

All values are average 5:10. Resuspensions in 6% Dextran.

TABLE III13.EFFECTS OF HEAT TRANSFER COATING AND TYPE OF CONTAINER ON RED CELL VIABILITY (PROCESS III)- System: BBC in suspending medium containing 60% w./v. Plasma-20% w./v. K-30 PVP-0.075 M N 9.01.

Dilution: BBC/Total vol.=1/2.6.

d N; 200 cycles/min, 2% in. ampl. Warming 37 0. water Conditions: 150 cycles/min.

All values a Cooling in liqui 2% in. ampl.

re averages :Ela'. Resuspensions in 6% Dextran. 

1. A PROCESS FOR FREEZING ERYTHROCYTES IN BULK QUANTITIES WHICH COMPRISES PROVIDING IN A CONTAINER A MIXTURE OF ERYTHROCYTES AND AN AQUEOUS MEDIUM CONTAINING AT LEAST ABOUT 10 WEIGHT PERCENT BASED ON THE WEIGHT OF THE MEDIUM OF A HIGH MOLECULAR WEIGHT, WATER SOLUBLE POLYMER EXTRACELLULAR PROTECTIVE ADDITIVE, IMMERSING SAID CONTAINER IN A REFRIGERANT BATH AT A TEMPERATURE NOT WARMER THAN ABOUT - 100*C. AND TUBULENTLY AGITATING SAID MIXTURE SUCH THAT THE RATE OF HEAT TRANSFER IS AT LEAST 14,000 B.T.U./(HR.) (SQ. FT. OF CONTAINER SURFACE) AND CONTINUING SUCH REFRIGERATIVE CONTACT FOR A SUFFICIENT DURATION TO FORM A FROZEN SHELL OF SAID MIXTURE ON THE INNER SURFACE OF SAID CONTAINER. 